Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Sox11 expression in astrocytic gliomas: correlation with nestin/c-Met/IDH1-R132H expression phenotypes, p-Stat-3 and survival.

BACKGROUND: Sox11 is a transcription factor expressed in foetal and neoplastic brain tissue, including gliomas. It has been shown to suppress the tumourigenicity of glioma stem cells in vivo, thereby being hypothesised to function as a tumour suppressor. METHODS: We investigated the expression of Sox11 in 132 diffuse astrocytomas in relation to the regulator cell marker nestin, c-Met and IDH1-R132H, which have shown to be differentially expressed among the molecular subgroups of malignant gliomas, as well as to an inducer of astrocytic differentiation, that is, signal transducer and activator of transcription (p-STAT-3), clinicopathological features and survival. RESULTS: Sox11 immunoreactivity was identified in all tumours irrespective of grade, but being correlated with p-STAT-3. Three out of seven cases showed partial Sox11 promoter methylation. In >50% of our cases neoplastic cells coexpressed Sox11 and nestin, a finding further confirmed in primary glioblastoma cell cultures. Furthermore, nestin, c-Met and IDH1-R132H expression differed among grade categories. Cluster analysis identified four groups of patients according to c-Met, nestin and IDH1-R132H expression. The c-Met/nestin high-expressor group displayed a higher Sox11 expression. Sox11 expression was an indicator of favourable prognosis in glioblastomas, which remained in multivariate analysis and validated in an independent set of 72 cases. The c-Met/nestin high-expressor group was marginally with shorter survival in univariate analysis. CONCLUSIONS: We highlight the importance of Sox11 expression as a favourable prognosticator in glioblastomas. c-Met/nestin/IDH1-R132H expression phenotypes recapitulate the molecular subgroups of malignant glioma.

The acute effect of Orlistat on dietary glycotoxins in diabetics and healthy women.

AIM: Advanced glycation end products (AGEs) formation is implicated in diabetic complications. Exogenous AGEs, namely glycotoxins, are present in certain foods and are absorbed from the gastrointestinal tract. Experimental data suggest that lifestyle interventions reducing their content in diet have beneficial effect. METHODS: Fourteen healthy (age: 42.14 +/- 12.38 years; body mass index [BMI]: 27.85 +/- 7.06 kg/m2) and ten women with type 2 diabetes (T2DM) (age: 48.70 +/- 9.31 years; BMI: 32.55 +/- 7.14 kg/m2) were enrolled in the study. A meal rich in AGEs was provided in a two-day protocol and on day 2, 240 mg of Orlistat were administered post-meal. RESULTS: On day 1, serum AGEs levels showed a rise at 3 hours post-meal compared to baseline values in both groups (controls: 12.2%; P<0.001), T2DM: 2.6%; P=0.013), but at 5 hours post-meal only in the controls (control: 12.2%; P<0.001); T2DM: 1.9%; P=0.075). On day 2 at 3 hours post-meal control values showed a rise of 3.1% (P=0.003); T2DM of 1.9% (P=0.013); at 5 hours post-meal rise for controls was 4.6% (P=0.012); and for T2DM was 1.8% (P=0.009). The corresponding rise was significantly lower on day 2 only in controls at 3 and 5 hours post-meal (P=0.003; P=0.05, respectively). CONCLUSIONS: Orlistat reduced the absorption of glycotoxins acutely and improved the metabolic profile in the control group, without an apparent beneficial effect in the diabetic group. The clinical significance of this observation should be further investigated in normal population, while in diabetics long-term studies may be required to demonstrate possible clinically significant effects.

Minichromosome maintenance proteins 2 and 5 in non-benign epithelial ovarian tumours: relationship with cell cycle regulators and prognostic implications.

Minichromosome maintenance proteins (MCM) have recently emerged as novel proliferation markers with prognostic implications in several tumour types. This is the first study investigating MCM-2 and MCM-5 immunohistochemical expression in a series of ovarian adenocarcinomas and low malignant potential (LMP) tumours aiming to determine possible associations with clinicopathological parameters, the conventional proliferation index Ki-67, cell cycle regulators (p53, p27(Kip1), p21(WAF1) and pRb) and patients' outcome. Immunohistochemistry was applied in a series of 43 cases of ovarian LMP tumours and 85 cases of adenocarcinomas. Survival analysis was restricted to adenocarcinomas. The median MCM-2 and MCM-5 labelling indices (LIs) were significantly higher in adenocarcinomas compared to LMP tumours (P<0.0001 for both associations). In adenocarcinomas, the levels of MCM-2 and MCM-5 increased significantly with advancing tumour stage (P=0.0052 and P=0.0180, respectively), whereas both MCM-2 and MCM-5 increased significantly with increasing tumour grade (P=0.0002 and P=0.0006, respectively) and the presence of bulky residual disease (P<0.0001 in both relationships). A strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (P<0.0001) as well as p53 protein (P=0.0038 and P=0.0500, respectively). Moreover, MCM-2 LI was inversely correlated with p27(Kip-1) LI (P=0.0068). Finally, both MCM-2 and MCM-5 were associated significantly with adverse patients' outcome in both univariate (> or =20 vs >20%, P=0.0011 and > or =25 vs <25%, P=0.0100, respectively) and multivariate (P=0.0001 and 0.0090, respectively) analysis. An adequately powered independent group of 45 patients was used in order to validate our results in univariate survival analysis. In this group, MCM-2 and MCM-5 expression retained their prognostic significance (P<0.0001 in both relationships). In conclusion, MCM-2 and MCM-5 proteins appear to be promising as prognostic markers in patients with ovarian adenocarcinomas.

Inflammatory and endothelial markers in women with polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) carry a pattern of cardiovascular risk factors. Endothelial dysfunction and chronic inflammation are early findings in the atherosclerotic process. The purpose of the study was to investigate the coexistence of active inflammation markers and endothelial dysfunction in young women with PCOS, and their relationship with metabolic and hormonal abnormalities of the syndrome. MATERIALS AND METHODS: Twenty-five young women with PCOS and 25 controls of similar age and body mass index (BMI) were studied. Endothelial function was assessed by flow-mediated dilatation (FMD) on the brachial artery and smooth muscle cells injury was excluded by nitrate-induced dilatation (NID). Plasma levels of endothelin-1 (ET-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and high sensitivity C-reactive protein (hsCRP) were measured. Hormonal and metabolic profiles were determined in both groups. RESULTS: Flow-mediated dilatation (FMD) was statistically lower in PCOS (P < 0.001), whereas nitrate-induced dilatation (NID) was similar within the two groups. Polycystic ovary syndrome (PCOS) had statistically higher levels of ET-1 (P = 0.03), sICAM-1 (P = 0.01), sVCAM-1 (P = 0.02) and hsCRP (P = 0.01). Furthermore FMD was statistically higher in PCOS population with hsCRP 1 mg L(-1) when compared with PCOS population with hsCRP > 1 mg L(-1) (P = 0.02). Flow-mediated dilatation (FMD) was negatively related to hsCRP (r = -0.512, P = 0.015); ET-1 was positively related to free androgen index (r = 0.27, P = 0.05) and negatively to sex hormone-binding globulin (r = -0.465, P = 0.022); sVCAM-1 was positively related to total testosterone (r = 0.431, P = 0.036); hsCRP was positively related to BMI (r = 0.647, P = 0.001), and negatively related to FMD (r = -0.512, P = 0.015), quantitative insulin sensitivity check index (QUICKI) (r = -0.499, P = 0.018), and MATSUDA index (r = -0.445, P = 0.038). CONCLUSIONS: The present study demonstrates that endothelial dysfunction coexists and is influenced by the presence of increased serum levels of inflammation and endothelial activation markers in young women with PCOS. These parameters appear to be interrelated with hyperandrogenaemia in this insulin-resistant population.

Evidence of improved serum fatty acid profile of postmenopausal women receiving atorvastatin and raloxifene.

Raloxifene and atorvastatin have been shown to reduce the risk of cardiovascular disease associated with postmenopausal status and it has been postulated that their effects may be partly mediated by favourable changes in serum lipids and fatty acid composition. In the present study, individual administration of either raloxifene (Group A) or atorvastatin (Group B) or both (Group C) was compared for a period of 3 months and their effects on total lipids and fatty acids composition was evaluated. Postmenopausal women receiving both raloxifene and atorvastatin showed significant changes in the majority of serum lipids with important reductions in total cholesterol (p < 0.001), triglycerides (p < 0.001), LDL-C (p < 0.001) and Apo B levels (p < 0.001). Phospholipids concentrations (p < 0.01) as well as Apo A-I were also significantly raised (p < 0.001). Furthermore, oleic acid (18:1) and linoleic acid (18:2) levels were significantly increased (p < 0.01 and p < 0.001 respectively) followed by a marked reduction in palmitic acid (16:0) and arachidonic acid (20:4) concentrations (p < 0.01 and p < 0.001 respectively). The results of the study indicate that the serum lipid and fatty acid composition in postmenopausal women is influenced by the combined treatment of raloxifene and atorvastatin and a further attempt to evaluate the significance of these results is discussed.

Metformin administration improves endothelial function in women with polycystic ovary syndrome.

OBJECTIVE: The aim of this study was to investigate the endothelial status in young women with polycystic ovary syndrome (PCOS), using a simple and easily reproducible hemodynamic method combined with a biological marker and to evaluate the effect of metformin treatment on these parameters. DESIGN: Descriptive clinical trial. METHODS: Forty young women, 20 with PCOS and 20 normal women of similar age and body mass index were studied. Metformin (1700 mg daily) was administered for 6 months to the PCOS group. The endothelium status and the metabolic and hormonal profile were studied in both groups, as well as after metformin, by flow-mediated dilatation (FMD) on the brachial artery and by measurements of plasma endothelin-1 (ET-1) levels. RESULTS: FMD was impaired in the PCOS group when compared with controls (3.24+/-0.71% vs 8.81+/-1.07% respectively, P<0.0001), but this difference normalized after metformin treatment (PCOS(post-metformin) vs controls: 8.17+/-1.26 vs 8.81+/-1.07%, P = 0.70) since the values significantly improved after metformin treatment (PCOS(pre-metformin) vs PCOS(post-metformin): 3.24+/-0.71 vs 8.17+/-1.26%, P=0.003). ET-1 levels were significantly higher in the PCOS women compared with the control group (7.23+/-0.50 vs 4.99+/-0.69 fmol/l, P=0.01), they improved significantly after metformin treatment (PCOS(pre-metformin) vs PCOS(post-metformin): 7.23+/-0.50 vs 3.57+/-0.60 fmol/l, P<0.0001) and their difference compared with the control group was reversed (PCOS(post-metformin) vs controls: 3.57+/-0.60 vs 4.99+/-0.69 fmol/l, P=0.13). Metformin administration improved hyperandrogenemia. However, in this study, mathematical methods used to assess insulin resistance failed to show any detected alteration after treatment with metformin. CONCLUSIONS: PCOS women were found to exhibit endothelial dysfunction compared with controls, which was reversed 6 months after metformin administration.

Evaluation of serum lipids and high-density lipoprotein subfractions (HDL2, HDL3) in postmenopausal patients with breast cancer.

Breast cancer patients are known to be at increased risk for developing other chronic diseases including cardiovascular disease. Studies by different investigators have shown a correlation between increased dietary fat or hypercholesterolemia and the occurrence of breast cancer. Since previous studies on lipoprotein subfractions in this type of cancer have been inconsistent, we evaluated the lipids and lipoprotein subfraction levels in postmenopausal patients with breast cancer in an attempt to identify the risk for the development of cardiovascular disease. The study included 132 patients, 56 of which were suffering from breast cancer, 32 from pancreatic and 44 age-matched controls. Total cholesterol (TC), triglycerides and lipoprotein fractions as well as TC/High density lipoprotein (HDL) and HDL2/HDL3 ratios were estimated by standard laboratory techniques. An increase in triglycerides and a decrease in HDL-cholesterol, especially in the HDL2 subfraction, were observed in patients with breast cancer as compared to the controls (P < 0.05). The maximum changes in TC, and HDL concentrations were observed in patients with advanced disease. Analysis of indexes of atherosclerosis (TC/HDL, and HDL2/HDL3 ratios) demonstrated that breast cancer patients had significantly higher TC/HDL ratio (6.44+/-1.24) compared with controls (3.43+/-0.57, p = 0.001), and patients with pancreatic cancer (3.79+/-0.15, p = 0.027). The results have demonstrated an unfavourable lipid profile in untreated breast cancer patients with high atherosclerosis indexes. This observation is of great importance, considering the potential use of endocrine therapy that could result in further deterioration of lipid indexes. We propose the evaluation and monitoring of lipid profile prior and after the induction of hormonal therapy in breast cancer patients, as a routine in clinical setting.

Effects of hemodialysis on serum lipids and phospholipids of end-stage renal failure patients.

Patients with chronic renal failure undergoing periodic maintenance hemodialysis frequently present dyslipoproteinaemia which has been linked to the sharply increased risk of cardiovascular disease in these subjects. Reported defects on lipoprotein-related enzyme activities suggest a possible influence of hemodialysis not just to plasma lipid and lipoprotein levels but also to the composition of cell membranes. In this study, it was investigated whether the reported lipid abnormalities are accompanied by changes in serum phospholipids levels. Blood samples were obtained from 140 patients undergoing maintenance hemodialysis treatment and 122 normolipidemic healthy controls and analyzed for total serum phospholipids and their individual subclasses, as well as for total cholesterol and triglycerides, HDL-cholesterol and its subclasses. A significant decrease was observed in serum HDL cholesterol levels (p < 0.001) and its subclasses, HDL2-cholesterol (p < 0.01) and HDL3-cholesterol (p < 0.01) in patients when compared with healthy controls. A critical increase in the serum triglyceride content (p < 0.001) of patients was also observed. In addition, the serum levels of sphingomyelin (p < 0.01) and diphosphatidylglycerol (p < 0.001) were increased in the patient group, while the levels of phosphatidylcholine (p < 0.01) and phosphatidylinositol (p < 0.01) were significantly decreased in the patient group compared to healthy controls. In conclusion, this work clearly demonstrates that hemodialysis treatment contributes significantly to the dyslipidemic profile of end-stage renal failure patients by altering serum lipoprotein and phospholipids concentrations.

Comparative analysis of oestrogen and raloxifene effects on the phospholipid composition of high density lipoproteins in healthy postmenopausal women.

The beneficial effect of selective oestrogen receptor modulators such as raloxifene in cardiovascular disease may be mediated partly by favourable changes in the phospholipid composition of high density lipoprotein (HDL) subclasses. In Group A (oestrogen alone) HDL2 phosphatidylcholine increased (P<0.001), while there was a decrease in HDL2 phosphatidylinositol (P<0.05) and HDL2 phosphatidylethanolamine (P<0.05) compared to controls (baseline). In the same group, HDL3 phosphatidylcholine increased (P<0.001) and HDL3 phosphatidylethanolamine decreased (P<0.01). In Group B (raloxifene) HDL2 phosphatidylcholine increased (P<0.001) as well as HDL2 diphosphatidylglycerol (P<0.01) while there were decreases in HDL2 sphingomyelin (P<0.01) and HDL2 phosphatidylethanolamine (P<0.05). In the same group, an increase in HDL3 phosphatidylcholine (P<0.001) and a reduction in HDL3 phosphatidylinositol (P<0.05) were observed as well as a decrease in HDL3 phosphatidylethanolamine (P<0.01) and HDL3 diphosphatidylglycerol (P<0.05). The significance of these results is discussed.

Design and synthesis of a multivalent homing device for targeting to murine CD22.

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail. and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohn's disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAc alpha-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper. the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAc alpha-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAc alpha-2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAc alpha-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAc alpha-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.

A structure-function study of ligand recognition by CD22beta.

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.